1. Field of the Invention
The present invention is directed to peptides that, in association with Class I MHC molecules, form epitopes recognized by cytotoxic T-cells specific for human diseases, to immunogens comprising said epitopic peptides, and to related compositions, methods and apparatus.
2. Description of the Background Art
There are two basic immune defenses in vertebrates. Humoral immunity is provided by antibodies, which defend primarily against bacterial, and the extracellular phase of viral, infections. Cellular immunity is provided by T cells, and their response is effective against microorganisms, parasites, cancer cells, foreign tissue (transplants), and both the intracellular and extracellular phase of viral infections. The cytotoxic T cell (lymphocyte) is a particular class of cellular immune response effector cell which eliminates invaders directly, e.g., by lysis. The two systems of cellular immunity interact in complex ways.
The present invention therefore contemplates the inhibition of diseases caused by viruses, bacteria, fungi, protozoa, algai, and other microorganisms, by multicellular parasites, and by cancer cells.
The prevention and treatment of cancer, and especially of melanoma, is of particular interest.
Melanoma affects 30,000 new patients per year in the United States. It is a cancer manifested by the unabated proliferation of melanocytes. Eighty percent of melanoma patients are diagnosed during their productive years between the ages of 25 and 65. The incidence of melanoma is rapidly increasing, in 1935 the lifetime risk of developing melanoma was 1:1,500 individuals, at present, the risk has risen to 1:105. It is believed that by the year 2000 the risk of developing melanoma will increase to about 1:70 to 1:90. Early diagnosis and treatment of this disease is crucial. Once a primary tumor becomes metastatic the disease is almost always fatal.
Cytotoxic lymphocyte (CTL) response has been shown to be an important host defense against malignant cells, Rock et al. J. Immunol., (1993), 150:1244.
Lymphocytes isolated from patients having melanoma, when stimulated in vitro with recombinant interleukin-2 (rIL-2) and autologous melanoma cells, develop a melanoma specific cytotoxic response, Vose et al., Nature, (1982), 296:359; Knuth et al., Proc. Natl. Acad. USA, (1984), 81:3511; Slingluff et al., Arch. Surg., (1987), 122:1407; Darrow et al., Cancer, (1988), 62:84; Slingluff et al., J. Natl. Cancer Inst., (1988), 80:1016; Slingluff et al., Ann. Surg., (1989), 210:194; Muul et al., J. Immunol., (1987), 138:989; Van den Eynde et al., Int. J. Cancer, (1989), 44:634; Anichini et al., Int. J. Cancer, (1985), 35:683. The majority of melanoma-specific effector lymphocytes are CD8+ cytotoxic T lymphocytes (CTL) that are restricted by class I Major Histocompatibility Complex (MHC) molecules, Vose et al; Slingluff et al (1988), supra, Hersey et al., Cancer Immunol. Immunother., (1986), 22:15. These characteristics are resent whether CTL have been generated from peripheral blood lymphocytes (PBL), lymph node cells, or tumor infiltrating lymphocytes.
The evidence that the CTL response to human melanoma is restricted by class I MHC molecules includes demonstration of cross-reactivity for allogenic melanoma cells that share a restricting class I MHC molecule with the autologous tumor. The HLA-A2 molecule and its variants, of which HLA-A2.1 is by far the most common, is an effective restricting element for the melanoma-specific CTL response. Additionally, melanoma-specific HLA-restricted CTL lyse the majority of A2+ melanomas tested, Darrow et al., J. Immunol., (1989), 142:3329; Wolfel et al., J. Exp. Med., (1989), 170:797; Hom et al., J. Immunother., (1991), 3:153. By demonstrating lysis of A2-melanomas transfected with the A2.1 gene, it has been shown that these transfected melanomas can present the epitopes recognized by A2-restricted melanoma-specific CTL, Kawakami et al., J. Immunol., (1992), 148:638. These results suggest that these CTL recognize A2-restricted epitopes that are shared by the majority of melanomas, although very little is known about the number and identity of their epitopes.
Class I molecules of the Major Histocompatibility Complex (MHC) bind to peptides derived from intracellular pathogens or from proteins expressed in tumor cells, and present them on the cell surface to the host immune system. The mechanism of peptide presentation involves protein synthesis and proteolysis in the cytosol, followed by transport of peptides into the endoplasmic reticulum (ER), through the action of the TAP transporter molecules. Peptides then become associated with newly synthesized class 1 molecules, and the resulting complexes move to the cell surface. Proteins that are membrane associated or secreted contain signal sequences that cause them to be contranslationally transferred into the ER from membrane-bound ribosomes. Such proteins would thus be protected from the action of cytoplasmic proteases. However, since peptide epitopes do arise from such proteins, although their TAP dependent expression is unclear, it has been assumed that the proteolysis to generate these peptide epitopes occurs after these proteins have been aberrantly translated on cytoplasmic ribosomes.
Adoptive transfer of tumor stimulated CTL has been associated with some tumor regressions, Rosenberg et al., N. Eng. J. Med., (1988), 319:1676.
An alternate approach to augmenting the T-cell response to melanoma is the use of a vaccine to stimulate CTL in vivo (active specific immunotherapy). Epitopes for CD8+ CTL are believed to be short, usually 9-residue peptides that bind to a cleft on the surface of the class I MHC molecule, Udaka et al., Cell, (1992), 69:989; VanBleek et al., Nature, (1990), 348:213; Falk et al., J. Exp. Med., (1991), 174:425. These peptides, generated from proteolysis of endogenous proteins in the cytosol, are transported to the endoplasmic reticulum, where they become associated with newly synthesized class I MHC molecules. They are then transported to the cell surface, Elliott et al., Nature, (1990), 3348:195. CTL epitopes have been reconstituted in vitro by allowing exogenous peptides to bind to MHC molecules on the cell surface of target cells, Townsend et al., Annu. Rev. Immunol., (1989), 7:601.
Several CTL epitopes have been identified which comprise cysteine, e.g., MLLAYLYCL (SEQ ID NO:66) (see Table A and Boon, et al., Eur. J. Immunol., 24:759-64 (1994)). However, prior to the priority application, it had not been suggested that it could be advantageous to replace these cysteines with non-cysteine residues. Indeed, there would have been concern that such replacement would adversely effect the interaction of the epitope with the class I MHC molecule or with the T cell receptor, or the specificity of the response to the epitope.
Meadows, et al., Immunity, 6:273-81 (1997), published after the priority date, discloses a nine residue H-Y specific CTL epitope FIDSYICQV (SEQ ID NO:67). It was discovered that cysteinylation of the cysteine affected recognition, improving recognition by one clone, and reducing recognition by another.
DiModugno, et al, J. Immunother., 20:431-6 (1997), published after the priority date, characterized certain cysteine-containing CTL epitopes (HLYQCQVV (SEQ ID NO:68) and CLTSTVLV (SEQ ID NO:69)) of the HER-2/neu gene product. DiModugno made Ser and Ala substitution mutants of CLTSTVLV (SEQ ID NO:69) to prevent dimenzation; there was no effect, good or bad, on the binding of the peptide to high affinity HLA-A2.1 molecules, and hence no motivation to perform such substitutions on other epitopes in the future.
The present invention is particularly directed to xe2x80x9ccysteine-depletedxe2x80x9d CTL epitopes, in which one or more cysteines not essential to eliciting the desired CTL response are replaced by another amino acid, especially Ala, Thr, Ser or Gly.
The present invention relates to immunogens which are capable of eliciting a disease-specific cytotoxic lymphocyte response in at least some individuals, which response is directed to peptide epitopes carried by those immunogens, and to the use of those immunogens in active specific immunotherapy and immunoprophylaxis against that disease. Melanoma is of particular interest.
These immunogens may be used as vaccines, in active specific immunotherapy. The immunogens may be administered directly or by gene therapy. The epitopic peptides may also be used to stimulate lymphocytes, the latter then being used for adoptive immunotherapy.
An antigen associated with a particular disease may elicit a CTL response, in which case the antigen must be processed into peptide fragments which are CTL epitopes. In some instances these native CTL epitope contains cysteine. If so, it is possible that intermolecular disulfide bonds will form between cysteines of two different epitopic peptides. If the epitope contains two or more cysteines, it is possible to form intramolecular disulfide bonds, too. Applicants have discovered that such disulfide bond formation can interfere with the CTL response.
One may reduce the total Cys to one, thus preventing intramolecular disulfide bond formation, or to zero, thereby also blocking intermolecular disulfide bonding.
In a preferred embodiment, the native CTL epitope is one other than DiModugno""s CLTSTVQLV (SEQ ID NO:78), which is a CTL epitope of a breast and ovarian tumor-associated antigen.
In other preferred embodiments, the cysteine depletion is applied to a cysteine-incorporating CTL epitope of the melanoma-associated antigens pMel-17 or tyrosinase.
A native tyrosinase CTL epitope of particular interest is the peptide Lys-Cys-Asp-Ile-Cys-Thr-Asp-Glu-Tyr (SEQ ID NO:19). We have discovered that substitution of Ser or Ala for Cys-2 in peptide KCDICTDEY (SEQ ID NO:19) increases the ability to reconstitute the epitope in vitro, probably by prevention of intramolecular disulfide bonds. The same principles apply to the related EKCDICTDEY (SEQ ID NO:37) and DAEKCDICTDEY (SEQ ID NO:10) sequences, and indeed to any Cys-containing CTL epitopes.
Additional embodiments of the present invention are described below. References to melanoma should be deemed to apply, mutatis mutandis, to other diseases, too.